Upload
others
View
8
Download
0
Embed Size (px)
Citation preview
Bioengineering ToolboxNow available to industry clients through Bioengineering Solutions
Ideas Robust CommercialProcesses
Idea(molecule)
PathwayPredictor
Gene Pipeline
PathwayPrototyping
HT Strain Engineering
Fermentation Engineering
SeparationsEngineering Product
Technology Platform
BioinformaticsMetagenomicsDirected evolution
TX-TLQSS
HT CloningGenome editing
13C-fluxome
Metabolome
Proteome
Transcriptome
Genome
Genomatica’s Bioengineering
Toolbox
Sector Independent
Bioprocess Engineering (TEA, Scale-Up / Scale Down, Tech Transfer)
Precision FermentationPathway PredictorOH
HOBDO
Scalable Kilo Piloting
1
SimPhenyTM
Bioengineering SolutionsKey Offerings
Integrated Strain/Process Dev.
Idea(molecule)
PathwayPredictor
Gene Pipeline
PathwayPrototyping
HT Strain Engineering
Fermentation Engineering
SeparationsEngineering Product
BioinformaticsMetagenomicsDirected evolution
TX-TLQSS
HT CloningGenome editing
13C-fluxome
Metabolome
Proteome
Transcriptome
Genome
Genomatica’s Bioengineering
Toolbox
Bioprocess Engineering (TEA, Scale-Up / Scale Down, Tech Transfer)
Precision FermentationPathway PredictorOH
HOBDO
Scalable Kilo Piloting
FeedstockEvaluationBioprocess Scale-up/Scale down Quantitative Small Scale
Strain Diagnostics Pathway Enzyme EngineeringEvaluation Studies
develop a predictive and quantitative small scale in vivo assay for strain evaluation
enable reliable process scale-up; improve strain and process robustness for successful technology transfer
enable new business opportunities or improve existing fermentation processes
assess new bioprocess ideas using techno-economic analysis and computational biology tools;
develop enzymes for improved in vivoperformance; such as for activity and specificity on non-natural substrates;
provides a ‘view inside the cell’ to gain deep understanding of metabolism using systems-based modeling and omicsProcess Model
TechnicalMetrics(TRY,OUR,impurities,recovery,wastes,etc.)
CostImpact$/ton
Variable 1
Variable 2
2
Bioengineering ToolboxDemonstrated on Numerous Organisms
EukaryotesProkaryotes
MammaliancellsHumancells
Modeling
Systems-based omics
ProveninyeastandGram+andGram- bacteria
Molecular/Micro Biology
EukaryotesProkaryotes
Mammaliancells
QSS
ProveninyeastandGram+andGram- bacteria
Fermentation
Experienceineukaryotesandprokaryotes;yeast,
Streptomyces
GENO Technology Platform
3
SimPhenyTM – High Performance Strains by DesignModeling and Simulation
4 Deepunderstandingofmetabolicnetworkandimpactofgeneticchanges
4 Providesanunderstandingofbyproductsandthecausesfortheirproduction(Forexample,isATPorredoxlimiting?)
4 Prioritizationofpathways,straindesignsandprocessengineeringstrategies
4 Analysisandinterpretationof“-omics”datainglobalnetworkcontext
4
System Biology Tools Enable High Performance Strains
4 RegularOmicsexperiments4 Networkanalysistools4 Statisticaldataanalysis
Iterativemulti-omicsprovidesvaluableinformationaboutcellularenvironment
Data-drivenhypothesisgeneration/decisions
Genomics
Transcriptomics• RNAseq• qPCR
• Productionandevolvedstrains• SNPs,ISelements,deletions,duplications
Proteomics• iTRAQ (global)• MRM(targeted)
Fluxomics• 13C-labeltracing• Computationaltools
Metabolomics
0 20 40 602
4
6
8
10x 105
time [h]
tota
l NAD
(H)
M1M2G1G2
0 20 40 600
2
4
6x 105
time [h]
NADH
0 20 40 601
2
3
4
5x 105
time [h]
NAD
0 20 40 600
1
2
3
time [h]
NADH
/NAD
0 20 40 600
1
2
3
4x 105
time [h]
tota
l NAD
P(H)
0 20 40 600
1
2
3x 105
time [h]
NADP
H
0 20 40 600
1
2
3
4x 104
time [h]
NADP
0 20 40 600
5
10
15
time [h]
NADP
H/NA
DP
• TriplequadLC-MS• Orbitrap - exactmass
5
Provides Key Insights into Metabolism
Systems Biology Example: Eliminating GBL
Two approaches to eliminate GBL by-product:1. Delete genes responsible for GBL
formation, deduced from transcriptomics
2. Identify and introduce new hydrolase
GBL
O Ohydrolase • GBL formed by cyclization of 4HB-CoA (C-yield loss)• Boiling point very close to BDO- expensive separation• GBL formation enzyme induced and spontaneous
0.0
100.0
200.0
300.0
400.0
500.0
600.0
700.0
800.0
879 1889 1889+hydrolase
mMBDO mMGBL
BDO
GBLNoGBL
KO hydrolase
4-HB 4-HB-CoA 4-HBal BDO
AldCat2 AdhHO
O
OHHO
O
SCoA HOO
HHO
OHorHOO
OHHO
O
SCoA HOO
HHO
OHor HOO
OHHO
O
SCoA HOO
HHO
OHor
Biological Solution to an Engineering Challenge
8
Ø 321kbregionspansdiversefunctionsincludingessentialgenesandproteasesØ RegionbordershavemobilegeneticelementsØ StrainstabilizedbyremovalofflankingISelementsØ Performancerescued,nogenomicinstabilityobservedinsubsequentlots
Example: Improving Robustness of Commercial Strain
Problem:Ø Anewlotof‘startercultures’forproductionstrainslatedforscale-upperformedpoorly
Approach:Ø WGS(MiSeq)revealedalargeduplicationeventinnewlotrelativetopriorlots
Genetic Instability Identified and Resolved in 5 Weeks
7
Genomatica Enzyme Engineering
Enzyme Structure
HTP in vitro
Screening
In vivo Screening
&Validation
• Quantitative small-scale assay
• Fermentation• Systems/Omics
• Predictive HT assays• 96/384-well• ~ 30,000 clones/day
• DNA synthesis to construct only desired mutations
• Outsource structure determination
• Rosetta enzyme modeling
• Substrate docking
Smart Library Design
Optimizing in vivo Performance of Pathway Enzymes
10
Demonstrated applications: Improve substrate specificity Switch cofactor specificityIncrease specific rate Improve enzyme stabilityLower product inhibition
Example: Improving Downstream Pathway Enzymes
4-HB 4-HB-CoA 4-HBal BDO
AldCat2 AdhHO
O
OHHO
O
SCoA HOO
HHO
OHorHOO
OHHO
O
SCoA HOO
HHO
OHor HOO
OHHO
O
SCoA HOO
HHO
OHor
• ImproveCat2BDOtolerance• Discovery/evolutionapplied• →20Xactivityin1MBDO
• ImproveAld stability• Discovery/evolutionapplied• →10Xactivity,nodegradation
Variant223848
Ald223848FermTime(h):
Ald
85.00
90.00
95.00
100.00
105.00
110.00
115.00
120.00
Parent Cat2* ALD* Cat2*+ALD*
BDO(g/L)
Titer@48hr
EvolvedCat2
EvolvedAld
EvolvedCat2+Ald
Parentenzyme
BDOTiterinFermentations
EvolvedCat2andAld:4Individually– noimprovement4Combined– 15%increaseintiter
Multiplestrainchangesoftenrequiredforimprovedperformance
Improvements in Activity, Tolerance and Stability Accomplished
11
strainscreening&evaluationtopredictlarge-scaleperformance
quantitativemetabolicprofiling,scale-downdiagnostics
systems-levelmetabolicphenotyping(omics)
QSS: Quantitative Small Scale Technology
smartdesign
information
cleanexperiment
Ü V Vgenomaticatechnology
commercialtools
customtools&modeling
Smart Design to Maximize Information at Optimal Throughput and Low Cost
12
0.70.80.91
1.11.21.31.4
BR QSS
StrainAStrainBStrainCStrainD
0.7
0.8
0.9
1
1.1
1.2
BR QSS
StrainAStrainBStrainCStrainD
2 2.5 3 3.5 4 4.5 5 5.50
10
20
30
time(h)
H16P
/H6P
2 2.5 3 3.5 4 4.5 5 5.50
10
20
30
time(h)
Cit/akg
QSS: Proprietary In-house Methods
Predictive
Rankable
SystemsBiologyReady
Relative Product Yield 12
34
12 34
12
34
12 34
Relative Byproduct Yield
01234567
Sp. Product Rate
0
2
4
6
8
10
Sp. Substrate Rate
0.00
0.10
0.20
0.30
0.40
0.50
Product Yield
Metabolomics
QSS
BR
QSS BR
0
1
2
3
4
5
0
30
60
90
120
150
ug/m
g to
tal
Proteomics
Correlation Between Bioreactor and QSS Scale
11
Scalable Bioprocessing: Robust Commercial Process DeliverySmaller, Cheaper, Better, Faster
• Full capability process lab, 6000 ft2
– 36 bioreactors, 2 L, 5 L, 30 L
– Wide range of unit ops for separations and purification
– Integrated LIMS (microbe-to-product)
• Quant small-scale fermentation– 98% carbon closure, 2% CVs
– Mimics large-scale environment
– 200+ process variables monitored
• 30 L piloting suite– Kilogram samples
– 80% of the data in 20% of the time/cost
– Full technology transfer services
Flexible, high-precision fermentation
Kilo-scale integrated piloting
Fermentation
HTST
S/LSeparations
Centrifugation30Lscale
Recovery/Purification
Microfiltration
Nanofiltration
Chromatography
SolventExtraction
Distillation
12
Example: Highly Predictable Scale-up/down
Robust performance at commercial-scale
• Fermentation performance across lab, pilot, and demo scales is highly consistent, enabling rapid lab-to-commercial scale development path • Average commercial-scale performance over ~50 campaign fermentations equivalent to demonstration-scale performance
for same strain (+/-2%)• Low variability in performance across ~50 campaign fermentations, indicates process robustness and predictability
Consistent scale-up to commercial
13,000L
Demonstration Scale
Commercial Scale
~ 50x scale-up
Campaign StrainTiter 98%Rate 104%Yield 100%
Average fermentation performance
( ~50 runs at commercial scale vs. average demo
scale)
0%
20%
40%
60%
80%
100%
120%
0 10 20 30 40 50
Batch No.
Percent average commercial-scale IRR'
0% 20% 40% 60% 80%
100% 120%
0 10 20 30 40 50
Batch No.
Percent average commercial-scale yield
Top 5 Fermentation RunsFermentation Run
Consistent performance across development scales
0%
30%
60%
90%
120%
2L 30L 13kL
BD
O, g
/L
(as
% o
f 2L)
0%
40%
80%
120%
160%
2L 30L 13kL
Rat
e, g
/L.h
r (a
s %
of 2
L)
0%
30%
60%
90%
120%
2L 30L 13kL
Yiel
d, g
/g(a
s %
of 2
L)
13
2013 Kirkpatrick Chemical Engineering Achievement Award
Integrated Solutions Example: Correlation Between O2 Supply and Yield
Ü Diagnostics,omics,strainengineeringrequiredtoincreaseBOTHrateandyieldunderlowO2 conditions
4LoweringO2 decreasesexcessCO2andincreasesyield- good
4LoweringO2 decreasesgrowth,rate,andBDOtiter- bad
BDOtiter(g/L)
BDOprodu
ctivity
(g/L/hr)NormalO2
LowerO2
Biom
ass(g)
BDOYield
%ofG
lctoExcessC
O2
CommercializationYieldTarget
BDO↓Growth↓
Rate↓
CO2 ↓ Yield↑
14
Integrated Approach to Improving Fermentation Performance
Reducing Flux Through CO2 Forming PathwaysGlucose
NADH NADH ATP NAD(P)H NADPH 1,4-BDO
PPpathway
CO2
acetyl-CoA
OAA
aKG
SSA 4HB
CO2
CO2
CO2
CO2
PEP
G6P
isocitrate
succinate
malate
zwf
sucCD
aceBAK
1. pentosephosphatepathway2. completeoxidativeTCAcycle3. glyoxylate shunt
DeletedthreeCO2 formingpathways:
• BDOdecreased• Growthratedecreased• ExcessCO2%increased!!
1. 13Cisotopetracingrevealeda“rogue”flux,convertingsuccinyl-CoAtosuccinate&formingexcessCO2.
2. ExcessCO2 pathwaysmayberequiredtosupplycofactors(ATP,NADPH,NADH)forgrowth&maintenance.
RogueFlux- complementsDsucCD andcompletesTCAcycle
Ü Thecellistellingusitneedsmoreofthesecofactors
Systems Biology Based Diagnostics Results in Key Discovery
Identifying and Reducing the “Rogue Flux (RF)”
4Potentialsuccinyl-CoAhydrolasecandidatesprioritizedfordeletionusingbioinformatics &omics dataOAA
aKG
CO2
CO2
isocitrate
malate
DsucCD
Rogueflux
fumarate
citrate
→InsufficientATP?NAD(P)H?=>Bothneeded
LowBDO(10g/L) Slowgrowth
TCA-impairedstrainsperformpoorly
4TCA-impairedstrains– poorBDOproductionandgrowth
X BDO↑
cyd+
Growth↑
Dcyd,pntAB OE
16
Critical solution identified based on systems-based understanding of metabolism
140 g/L
2012/13 (650 m3)Commercial scale
2011Shipping tons at a time
2011 (13 m3)Integrated demo plant
2010 (3 m3)Piloting
2009 (30 L) Purified Bio-BDO
2008 (2 L)Bio-BDO producing organisms
GENO BDOTM Process
5 mg/L
50 g/L
80 g/L
Genomatica’s Capabilities: Geno BDO™ CommercializationProven in Practice: 5 years from Concept to Commercialization
20 g/L
100 g/L
17